The long range objective of the project is to broadly advance biomedical research by enhancing the performance of high performance liquid chromatography (HPLC) in determinations of drugs and other biologically relevant compounds. HPLC has found tremendous use in biomedical research; however, there are limitations to its effectiveness in separating pharmaceuticals and other compounds containing basic functional groups. Interaction of basic constituents with acidic silanol (SiOH) sites on siliceous substrates results in non-ideal separations marked by peak tailing and band broadening that hinders quantification. Problems also exist with the lack of reproducibility of HPLC columns and the subsequent need to undergo method development when columns are replaced. This project aims to advance the current understanding of the surface composition and chemistry of stationary phase materials for HPLC and to utilize this knowledge in the development of innovative bonded-phase substrates with improved HPLC performance. The specific aims of the research are four-fold. First, the project aims to study the surface composition of traditional (C8 and C18) and newly developed (e.g. polar embedded phases, monolithic columns, etc.) reversed-phase substrates for HPLC. The second aim of the research is to study and optimize the experimental conditions for the silylation reaction with silica to produce substrates with maximum silane loading, minimal, accessible silanol sites, and uniform ligand coverage. Third, the project aims to study the progressional changes and kinetics of the alkylsilylation of silica. Information gained through the third specific aim will contribute to the understanding of how the modification process occurs so that strides can be made in the development of synthetic approaches. The fourth specific aim is to study the performance of wellcharacterized silica substrates in standardized separations of pharmaceuticals and to correlate this data with knowledge gained regarding surface constituents of the respective bonded-phase materials. Two analytical techniques will be employed to provide the surface characterization studies; the proven and respected method of diffuse reflectance Fourier transform infrared spectroscopy (DRIFTS), as well as a new and powerful technique for structural elucidation, atomic force microscopy (AFM). AFM has tremendous potential for detailed, nanoscale surface analysis under a wide range of operating conditions, and as such, should complement well the information gained by the versatile DRIFTS technique. Advances made through completion of the research will ultimately enhance progress in all areas of biomedical research including studies related to AIDS, diabetes, heart disease, cancer, and many other conditions affecting health and the quality of life.